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WO2000003000A2 - Milieu exempt de serum pour mettre en culture des cellules animales - Google Patents

Milieu exempt de serum pour mettre en culture des cellules animales Download PDF

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Publication number
WO2000003000A2
WO2000003000A2 PCT/JP1999/003735 JP9903735W WO0003000A2 WO 2000003000 A2 WO2000003000 A2 WO 2000003000A2 JP 9903735 W JP9903735 W JP 9903735W WO 0003000 A2 WO0003000 A2 WO 0003000A2
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WO
WIPO (PCT)
Prior art keywords
serum
cells
protein hydrolysate
medium
free medium
Prior art date
Application number
PCT/JP1999/003735
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English (en)
Other versions
WO2000003000A3 (fr
Inventor
Kazushi Shibuya
Masaru Atsumi
Shigeyuki Tsunakawa
Kaneo Nogaki
Thomas Reid Fletcher
Katsuyuki Imada
Bjorn Kenneth Lydersen
Original Assignee
Chugai Seiyaku Kabushiki Kaisha
Irvine Scientific Sales Company, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chugai Seiyaku Kabushiki Kaisha, Irvine Scientific Sales Company, Inc. filed Critical Chugai Seiyaku Kabushiki Kaisha
Priority to EP19990960681 priority Critical patent/EP1097194A2/fr
Priority to AU63110/99A priority patent/AU6311099A/en
Priority to JP2000559222A priority patent/JP3934877B2/ja
Publication of WO2000003000A2 publication Critical patent/WO2000003000A2/fr
Publication of WO2000003000A3 publication Critical patent/WO2000003000A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0043—Medium free of human- or animal-derived components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/74—Undefined extracts from fungi, e.g. yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/76—Undefined extracts from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention relates to a serum-free medium for culturing animal cells, a method for culturing animal cells using the medium, and a method for producing a substance by culturing animal cells in the medium.
  • mammal-derived cells are used as host cells for recombinant production of human-derived proteins, etc. to be used in pharmaceuticals.
  • Some of mammal-derived cells are used as host cells.
  • One of the most commonly used mammal-derived cells is Chinese hamster ovary (CHO) cells.
  • selection markers methods for selecting recombinant cell clones using the markers and host cells which are necessary for application of recombinant DNA techniques, genetic deficiencies against which various selection markers function effectively and a large number of sub-cell lines exhibiting auxotrophy associated with such genetic deficiencies have been established.
  • a medium containing serum or protein components separated from serum has been commonly used for culturing mammal-derived cells.
  • a serum-free medium has been positively developed in order to eliminate contaminants in serum such as viruses or pathogenic prions which must not remain in final products, e.g. recombinant proteins.
  • a serum-free medium now under development is a basal medium supplemented with various peptide hormones and growth factor proteins for the induction or promotion of cell growth.
  • the basal medium contains appropriate quantities of the substances composing the transformed cells to be cultured (specifically, raw materials such as amino acids, precursors of nucleic acids or nucleosides, aliphatic acids, etc. to be used in the biosynthesis of various proteins, peptides, lipids, nucleic acids, etc.
  • supplementary components to maintain the cultivation rate at a high level is under review.
  • addition of supplementary components to promote the production of a gene product of interest, notably recombinant protein or the like, in the transformed cells in culture or to maintain such production at a high level is under review. It is expected to propose a serum-free medium having a cultivation ability comparable to that of the conventional serum-containing medium through selection of the above-mentioned supplementary components to be added to the medium and selection of the optimum amounts of addition of such components.
  • a new proposal of a serum-free medium is awaited which can achieve an ability comparable to that of the conventional serum-containing medium in culturing animal-derived cells, especially CHO cells, commonly used in the recombinant production of human-derived proteins for use m pharmaceuticals. Furthermore, proposal of a method for culturing animal cells, especially CHO cells, using such a new serum- free medium, as well as a method for performing recombinant production of a human-derived protein of interest at a high efficiently by such culturing is also desired.
  • the present invention has been made to solve the above problems. It is an object of the invention to provide a serum-free medium for culturing animal cells.
  • the present inventors have made extensive and intensive researches. As a result, it was found that growth of animal cells, in particular CHO cells, can be achieved in a serum-free medium which is obtained by adding various peptide hormones and growth factor proteins for induction or promotion of cell growth to a basal medium containing appropriate quantities of various components taken up by the cells from the outside and used as cell const ltuents / component s P e r s e . Subsequently, the inventors have optimized the composition of the basal medium in order to maintain the growth rate at a high level .
  • the growth rate of animal cells was still significantly inferior to the growth rate achieved in the serum-free medium described above supplemented with serum or serum- derived proteins, proteolysates , peptides, etc.
  • the present inventors have further found out that a cultivation ability comparable to the growth rate achieved in serum-containing media can be achieved in a serum-free medium by replacing the components of serum- containing media separated from animals (such as serum or serum- derived proteins, proteolysates, peptides) with a specific combination of components derived from plants and components derived from microorganisms which do not exhibit pathogenicity against human or mammals and by selecting optimum amounts of addition of such components. Based on these findings, the present invention has been achieved .
  • the present invention provides a serum-free medium for culturing animal cells, containing soybean protein hydrolysate and yeast extract.
  • the serum-free medium of the invention may further comprise wheat protein hydrolysate.
  • animal cells can be cultured without addition of components separated from animals, such as serum or serum-derived proteins, proteolysates, peptides.
  • the soybean protein hydrolysate may be added at 1-5 g per liter of the medium.
  • the yeast extract may be added at 1-5 g per liter of the medium.
  • wneat protein hydrolysate may be added at a rate of 0.5-3 g per liter of the medium.
  • the ratio by weight of the amount of addition of soybean protein hydrolysate to the amount of addition of yeast extract may be in the range from 80:20 to 60:40.
  • the amount of addition of wheat protein hydrolysate may come within the range from 5 to 60% of the total weight of the soybean protein hydrolysate and the yeast extract added.
  • animal cells preferably mammalian cells, more preferably Chinese hamster ovary (CHO) cells.
  • the animal cells may be transformed cells into which a foreign gene has been transferred.
  • the present invention also provide a method for culturing animal cells, comprising a step of culturing animal cells in a serum-free medium containing soybean protein hydrolysate and yeast extract.
  • the animal cells are preferably mammalian cells, more preferably CHO cells.
  • the animal cells may be transformed cells into which a foreign gene has been transferred.
  • the present invention provides a method for producing a substance, comprising a step of culturing animal cells in a serum-free medium containing soybean protein hydrolysate and yeast extract to thereby allow the animal cells to produce the substance and secrete it out of the cells and a step of isolating the substance from the serum-free medium.
  • the substance may be a protein or peptide.
  • the animal cells may be transformed cells into which a foreign gene has been transferred.
  • the substance produced by and secreted out of the animal cells may be a gene product from the transferred gene, e.g., a recombinant protein or peptide.
  • the animal cells are preferably mammalian cells, more preferably CHO cells.
  • Fig. 1 is a graph showing the effect of the amounts of addition of soybean protein hydrolysate and yeast extract on the growth rate of transformed CHO cells.
  • Fig. 2 is a graph showing the effect of the amounts of addition of soybean protein hydrolysate and yeast extract on the viability of transformed CHO cells.
  • Fig. 3 is a graph showing the effect of the amounts of addition of soybean protein hydrolysate and yeast extract on the yield of a recombinant protein by transformed CHO cells.
  • Fig. 4 is a graph showing the effect of addition of wheat protein hydrolysate as well as soybean protein hydrolysate and yeast extract on the growth rate of transformed CHO cells.
  • Fig. 5 is a graph showing the effect of addition of wheat protein hydrolysate as well as soybean protein hydrolysate and yeast extract on the viability of transformed CHO cells.
  • Fig. 6 is a graph showing the effect of addition of wheat protein hydrolysate as well as soybean protein hydrolysate and yeast extract on the yield of a recombinant protein by transformed CHO cells.
  • Figs. 7a and 7b are graphs showing the effect of the amount of addition of insulin on the growth rate of transformed CHO cells when subcultured in the presence of wheat protein hydrolysate in the medium in addition to soybean protein hydrolysate and yeast extract.
  • Figs. 8a and 8b are graphs showing the effect of the amount of insulin added on the yield of a recombinant protein by transformed CHO cells when subcultured in the presence of wheat protein hydrolysate in the medium in addition to soybean protein hydrolysate and yeast extract.
  • Fig. 9 is a graph showing the effect of the amount of addition of insulin on the viability of transformed CHO cells when subcultured in the presence of wheat protein hydrolysate in the medium in addition to soybean protein hydrolysate and yeast extract.
  • Fig. 10 is a graph comparing the yields of a recombinant protein by transformed CHO cells when the glucose contained in the medium is replaced with fructose for culturing in the presence of wheat protein hydrolysate in the medium in addition to soybean protein hydrolysate and yeast extract.
  • Fig. 11 is a graph comparing the addition of three components, soybean protein hydrolysate, yeast extract and wheat protein hydrolysate, with the addition of animal-derived proteins, bovine serum albumin, fetuin, human transferrin and primatone, as to the effect on the yield of a recombinant protein by transformed CHO cells.
  • Fig. 12 is a graph comparing the addition of three components, soybean protein hydrolysate, yeast extract and wheat protein hydrolysate, with the addition of animal-derived proteins, bovine serum albumin, fetuin, human transferrin and primatone, as to the effect on the viability of transformed CHO cells.
  • Fig. 13 is a graph comparing the addition of three components, soybean protein hydrolysate, yeast extract and wheat protein hydrolysate, with the addition of animal-derived proteins, bovine serum albumin, fetuin, human transferrin and primatone, as to the effect on the growth rate of transformed CHO cells.
  • the serum-free medium of the invention may be used for culturing animal cells, preferably mammalian cells, and more preferably CHO cells.
  • serum-free medium as used herein means a medium which does not contain serum or any serum components such as proteins separated from serum. Furthermore, the serum-free medium of the invention does not contain any components separated from animals, to say nothing of the above-mentioned serum or serum components such as proteins separated from serum.
  • Animal cells which can oe cultured in the serum-free medium of the invention included: mammal cells such as CHO cells, HeLa cells, baby hamster kidney (BHK) cells, rodent myeloma cells; insect cells such as D r o s o p h i l a cell lines; and transformed cells obtained by transferring a foreign gene into these cells.
  • mammal cells such as CHO cells, HeLa cells, baby hamster kidney (BHK) cells, rodent myeloma cells
  • insect cells such as D r o s o p h i l a cell lines
  • transformed cells obtained by transferring a foreign gene into these cells included: mammal cells such as CHO cells, HeLa cells, baby hamster kidney (BHK) cells, rodent myeloma cells; insect cells such as D r o s o p h i l a cell lines; and transformed cells obtained by transferring a foreign gene into these cells.
  • the serum-free medium of the invention contains, as its basal medium components, specific amounts of various nutrient components which are selected from inorganic substances, synthetic substances and plant-derived components and which are taken up from the outside by the animal cells to be cultured and used as cell constituents/components P e r s e , and specific amounts of synthetic or recombinant peptide hormones or cell growth factors which directly act on the animal cells to induce or promote their cell growth.
  • the medium of the invention contains soybean protein hydrolysate and yeast extract as supplementary components. More preferably, wheat protein hydrolysate is also added to the medium of the invention.
  • the serum-free medium of the invention may be free from components separated from mammals.
  • the components separated from mammals mean not only serum but also proteins separated from serum (e.g., serum albumin, transferrin, fetuin, various peptide hormones, growth factor proteins), proteins and peptides separated from mammals such as blood-containing tissues (e.g., beef hydrolysate), and organic components separated from mammalian sources (e.g., lipids and carnohydrates ) and so on.
  • the various nutrient components to be added to the serum-free medium of the invention as basal medium components include the components mentioned in Table 1 below. These components are taken up from the outside by the animal cells (preferably mammal cells, more preferably CHO cells) to be cultured and used as cell constituents/com ponents p e r s e , and they are selected from inorganic substances, synthetic substances and plant-derived components.
  • the basal medium components include various amino acids, nuclear acids or precursors thereof, essential metals and inorganic ions, lipids, vitamins, coenzy es and cofactors , organic substances to be used as energy sources, carbon sources and nitrogen sources. These components are used during the process of biosynthesis of various biological substances constituting the animal cells.
  • the basal medium components further include various peptide hormones and growth factor proteins that are not directly separated from animals, i.e., those which are produced with recombinant techniques, synthesized artificially or separated from plants.
  • various buffer components for maintaining the pH at a desired level and inorganic osmotic pressure adjustors for controlmg the osmotic pressure at a desired level are also added to the medium of the invention.
  • Basal Medium Composition A Representative Example
  • an iron-EDTA complex is also used as an iron source in addition to an inorganic iron salt.
  • the amount of addition of this complex represents the final concentration in the medium when the preparation of the medium has been completed.
  • the basal medium composition described above sufficiently contains various nutrient components which are used by the cells in the oiosynthesis of necessary substances for constituting the cells, e.g., cell membrane, nuclear membrane, peptides, proteins, various DNAs and RNAs.
  • These nutrient components include essential and non-essential amino acids necessary for CHO cells; monosaccha ⁇ des to be used as carbon sources or energy sources; various nucleic acids and vitamins; lipids and aliphatic acids necessary for CHO cells; electrolyte ions, metal ions and acids to be contained in the cytoplasm; and various metal elements and co-enzymes to be used by enzyme proteins.
  • the nutrient components described above are taken up and absorbed by the cells from the medium, and used for the maintenance and division of the cells. Further, buffer components to retain the pH of the medium at an appropriate level for the cultivation and osmotic pressure adjusting components to maintain an appropriate osmotic pressure are also contained in proper amounts.
  • the pH is adjusted in the range from 7.0 to 7.5 and the osmotic pressure in the range from 280 to 320.
  • the amounts of NaHC0 3 , etc. (buffer components) and the amounts of NaCl, etc. (osmotic pressure adjusting components) in the medium are adjusted so that the pH of the medium comes within the range from 7.2 to 7.4 and the osmotic pressure within the range from 290 to 300.
  • composition shown in Table 1 is just one representative composition of the basal medium which may be used in the medium of the invention and one or more of the components listed in Table 1 may be replaced with equivalent components.
  • two substances equivalent with each other such as amino acids cysteme and cystine, may be interchanged.
  • salts other than those listed in Table 1 may be used as long as they are water- soluble inorganic salts and can be used in a medium for culturing animal cells.
  • a part of glucose which is commonly used as a monosaccha ⁇ de may be replaced with fructose.
  • Certain vitamins such as vitamin Bi 2 consist of several compounds that have similar actions and are interchangeable. A part of such vitamins may be replaced with such equivalent compounds.
  • peptide hormones and growth factor proteins which are added to the serum-free medium of the invention so that they act directly on animal cells to thereby induce or promote cell growth
  • synthetic or recombmantly produced hormones and proteins are used. This means that those which are synthesized or recombmantly produced and sufficiently purified that they are free from unwanted contamination as with virus, mycoplasma, pathogenic prion, etc. should be used.
  • Peptide hormones or cell growth factors which were already reported to act on animal cells directly to thereby induce or promote cell growth have been added to a medium conventionally. They may be used similarly in the present invention.
  • peptide hormones or cell growth factors which induce or promote cell growth include recombinant insulin, recombinant insulins having a modified amino acid sequence and exhibiting a physiological activity comparable to that of natural insulin, and recombinant insulin-like growth factor.
  • recombinant insulins a recombinant human insulin may be used.
  • a commercial recombinant human insulin such as nucellin (product name) may be used.
  • soybean protein hydrolysate yeast extract and wheat protein hydrolysate which characterize the serum- free medium of the invention most will be described.
  • these three supplementary components are not essential components for culturing animal cells, they are effective in maintaining at high level the rate of the cell growth induced or promoted by the above- mentioned peptide hormones or cell growth factors, or in further increasing the growth rate.
  • these three components are effective in promoting the production of a recombinant protein or peptide of interest which is achieved by culturing recombinant animal cells, or in maintaining the production rate at high level.
  • cultivation of cells can be achieved with the basal components alone (i.e., the serum-free medium of the invention without soybean protein hydrolysate , yeast extract and wheat protein hydrolysate) though the cultivation rate is low.
  • the technical significance of the serum-free medium of the invention is that appropriate amounts of soybean protein nydrolysate, yeast extract and wheat protein hydrolysate are added as supplementary components to the basal medium components to thereby achieve a higher cultivation efficiency. In view of the effect of these three components, they are regarded as supplementary components to the basal medium.
  • the component which mainly contributes to maintain the growth rate at high level or further improve the growth rate is soybean protein hydrolysate; and the component which mainly contributes to maintain the production rate of a recombinant protein or peptide of interest at high level or the promote such production is yeast extract.
  • soybean protein hydrolysate is effective in reducing the mortality of the recombinant animal cells during production of a recombinant protein or peptide of interest.
  • wheat protein hydrolysate manifests its effect well when added to the medium together with soybean protein hydrolysate and yeast extract, rather than added to the medium alone.
  • the soybean protein hydrolysate used in the invention may be a soybean protein hydrolysate obtained from soybean of any quality.
  • a commercial soybean protein hydrolysate used for culturing microorganisms e.g., DMVSE50MK (DMV), DMVSE50MAF (DMV), HyPep 1601 (Quest), Soy Protein Hydrolysate: Hysoy (Quest), or the like is used.
  • Soybean protein hydrolysate may be converted to a soluble polypeptide by partial hydrolysis with various enzymes such as digestive enzymes.
  • Yeast extract is also available as commercial products for use in culturing microorganisms.
  • a commercial yeast extract such as HyYeast 455 (refined yeast extract; Quest), Springer Yeast Extract UF10 (Bio Springer), Fermax 5902AG (Red Star) or the like is used.
  • Yeast extract is a material which is obtained by crushing dry yeast and separating the intracellular soluble fraction by extraction.
  • Yeast extract is available as a material containing various coenzymes and cofactors.
  • the wheat protein hydrolysate used in the invention may be a wheat protein hydrolysate obtained from wheat of any quality.
  • a commercial wheat protein hydrolysate used for culturing microorganisms e.g., HyPep 4402 (Quest) is used.
  • Wheat protein hydrolysate may be obtained as a soluble polypeptide by partially hydrolyzing protein as in wheat embryos with various digestive enzymes .
  • Soybean protein hydrolysate, yeast extract and wheat protein hydrolysate are commercially available in various forms, and it is convenient to use such commercial products. It is particularly preferable to use commercial products intended for culturing microorganisms.
  • the amounts of addition of these components are expressed in dry weight.
  • the amount of addition of soybean protein hydrolysate is selected depending on the animal cell density at tne beginning of cultivation.
  • the amount is selected from the range of at least 1 to 6 g/liter; usually from the range of 1 to 5 g/liter; preferably from the range of 2 to 4 g/liter.
  • the amount of addition of yeast extract is selected depending on the rate of rise of the animal cell density as a result of the cultivation.
  • the amount is selected from the range of at least 0.5 to 5 g/liter; usually from the range of 1 to 5 g/liter; preferably from the range of 1 to 3 g/liter.
  • the ratio (by weight) of the amount of addition of soybean protein hydrolysate to the amount of addition of yeast extract is preferably at least 50:50, more preferably at least 60:40.
  • the amount of addition of yeast extract is unduly small, the effectiveness in promoting the production of a recombinant protein in cultured cells is insufficient.
  • the ratio (by weight) of the amount of addition of soybean protein hydrolysate to the amount of addition of yeast extract is preferably not more than 90:10, more preferably 80:20 or less. Considering these two conditions together, the ratio (by weight) of the amount of addition of soybean protein hydrolysate to the amount of addition of yeast extract is preferably in the range of 50:50 to 90: 10, more preferably in the range of 60:40 to 80:20.
  • wheat protein hydrolysate is effective in improving the cultivation rate and reducing the cell death resulting from the production of a recombinant protein or peptide by the cultured cells.
  • the amount of addition of wheat protein hydrolysate is selected considering the above-mentioned amounts of addition of soybean protein hydrolysate and yeast extract. In particular, it is preferable to select this amount depending on the amount of addition of yeast extract.
  • the amount of addition of yeast extract is in the range from 1 to 5 g/ liter
  • the amount of addition of wheat protein hydrolysate is preferably in the range from 0.5 to 3 g/liter.
  • the amount of addition of wheat protein hydrolysate is more preferably in the range from 0.5 to 2 g/liter, particularly preferably around 1 g/liter.
  • the amount of addition of wheat protein hydrolysate is selected preferably from the range of 5 to 60%, more preferably from the range of 10 to 40%, of the total weight of the soybean protein hydrolysate and the yeast extract added.
  • the amount of addition of soybean protein is selected from the range of 2 to 4 g/ liter and the amount of addition of yeast extract is selected from the range of 1 to 3 g/liter, it is more preferable to select the amount of addition of wheat protein hydrolysate from the range of 10 to 40% of the total weight of the soybean protein hydrolysate and the yeast extract added.
  • the promotion and maintenance of cultivation rate and the promotion of the production of a recombinant protein or peptide by the cultured cells are achieved by adding soybean protein hydrolysate and yeast extract in the preferable amounts stated above.
  • the effects of these two components can be further increased by also adding wheat protein hydrolysate in the preferable amount stated above.
  • a particular advantage to the promotion and maintenance of cultivation rate results from the addition of wheat protein hydrolysate as well as the above two components since this is effective in widening the allowable range of the amounts of addition of peptide hormones and cell growth factors for inducing and promoting the cell growth.
  • the amount of addition of recombinant insulin as the above-mentioned peptide hormone/cell growth factor to induce and promote the cell growth is usually 5 mg/liter; this amount can be reduced to 1 mg/liter while maintaining a sufficient cultivation rate, if wheat protein hydrolysate is added to the medium in addition to soybean protein hydrolysate and yeast extract.
  • the serum-free medium of the invention can be prepared by adding each of the above-described components to an appropriate amount of water, dissolving or suspending them, homogenizing the components in the resultant medium, and finally adding a small amount of water to the medium to make a specified volume.
  • the components to be added to the serum-free medium of the invention are chiefly selected from inorganic substances, synthetic substances, plant-derived components, plant-derived protein hydrolysates, and recombinant proteins or peptides produced with recombinant DNA techniques .
  • a part of the ammo acids to be added to the medium may be such am o acids that are synthesized from starting materials by an enzyme reaction using a microorganism or an enzyme it produces and then purified.
  • Animal cells preferably mammal cells, more preferably CHO cells can be cultured using the serum-free medium of the invention containing soybean protein hydrolysate, yeast extract and, optionally, wheat protein hydrolysate. Specific examples of the animal cells are as described previously.
  • a method of culturing CHO cells for example, cells are seeded in the medium at an initial density of 1-5 x 10 s cells/ml, preferably 2-4 x 10 5 cells/ml and cultured at 37 °C under 5% C0 2 .
  • the cultivation method using tne serum-free medium of the invention is applicable to transformed CHO cells for use in the production of various recombinant proteins or peptides.
  • recombinant protein or peptide produced by culturing such transformed CHO cells include human t-PA, human immune interferon ⁇ , human interferon ⁇ , granulocyte colony stimulating factor (G-CSF), granulocyte macrophage colony stimulating factor (GM- CSF), erythropoiet , interleukins such as IL-1, IL-6, urok ase, albumin, blood coagulation factor VIII, and recombinant antibodies such as a humanized anti-human IL-6 receptor antibody.
  • G-CSF granulocyte colony stimulating factor
  • GM- CSF granulocyte macrophage colony stimulating factor
  • erythropoiet interleukins
  • interleukins such as IL-1, IL-6, urok ase, albumin, blood coagulation factor VIII
  • recombinant antibodies such as a humanized anti-human IL-6 receptor antibody.
  • the serum-free medium of the invention a method for culturing CHO cells using the medium, and a method for producing a recombinant protein by culturing transformed CHO cells are described with reference to specific examples.
  • a humanized anti-human IL-6 receptor antibody-producing transformed CHO cell clone is used as the transformed CHO cell, and a system to produce the humanized anti-human IL-6 receptor antibody as the recombinant protein is described.
  • the serum-free medium of the invention can be used for culturing not only those transformed CHO cells which are obtained by using DHFR gene-deflcient CHO cell clone as a host cell and DHFR gene as a selection marker, but also other transformed CHO cells which are obtained by using other selection marker such as glutamine synthetase gene. In the latter case, the serum-free medium of the invention can also achieve an extremely efficient cell growth and stabilization thereof .
  • a humanized anti-human IL-6 receptor antibody-producing transformed CHO cell clone was obtained by transferring into a host cell a DHFR gene-deflcient CHO cell clone [a dihydrofolate reductase- deficient clone (DXB11 cells) separated from KI clone after treatment with 3 H-deoxyu ⁇ dine; described in L.H. Grof , L.A. Chasm, Mol . Cell Biol .
  • an expression vector was introduced into DXB11 cells by electroporation . Thereafter, colonies surviving in a selection medium (containing bovine serum but not containing nucleotides) were selected.
  • a selection medium containing bovine serum but not containing nucleotides
  • soybean protein hydrolysate and yeast extract can achieve a remarkable improvement in the cultivation rate of transformed CHO cells and in the yield of the recombinant protein produced by the cultured cells.
  • the correlation between the improvement and the amounts of addition of soybean protein hydrolysate and yeast extract was also examined.
  • soybean protein hydrolysate a commercial product HySoy (Quest) was used, and as yeast extract a commercial product UF10 (Bio Springer) was used.
  • the soybean protein hydrolysate and the yeast extract were added to the basal medium composition shown in Table 2 below to prepare a serum-free medium.
  • the total amounts of addition of the soybean protein hydrolysate and the yeast extract was 5 g/liter. While varying the ratio (by weight) of the amount of addition of soybean protein hydrolysate to the amount of addition of yeast extract, cell counts on day 4 of the cultivation and thereafter, cell viabilities from day 4 to day 7, and the total amounts of the recombinant protein accumulated in the medium up to day 7 were compared.
  • an iron-EDTA complex is also used as an iron source in addition to an inorganic iron salt.
  • the amount of addition of this complex represents the final concentration in the medium when the preparation of the medium has been completed.
  • the culture conditions were as follows. Thirty milliliters of the medium was placed in a 125 ml flask. Cells were seeded at a density of 3.0 x 10 5 cells/ml and cultured under agitation at 160 rpm, at 37 °C , under 5% C0 2 . The results, i.e., cell count on day 4 of the cultivation and thereafter, cell viability from day 4 to day 7, and the total amount of the recombinant protein accumulated in the medium up to day 7 are shown in Figs. 1, 2 and 3, respectively.
  • wheat protein hydrolysate as well as soybean protein hydrolysate and yeast extract
  • the medium can achieve not only a remarkable improvement in the cultivation rate of transformed CHO cells and in the yield of the recombinant protein produced by the cultured cells, but also a considerable inhibition of the decrease in cell viability resulting from the production of the recombinant protein in the cultured cells.
  • the amount of addition of wheat protein hydrolysate suitable for inhibiting the decrease in cell viability, and the optimum ranges of the amounts of soybean protein hydrolysate and yeast extract to be added simultaneously and the ratio thereof were examined.
  • As wheat protein hydrolysate a commercial product HyPep 4402 (Quest) was used.
  • Soybean protein hydrolysate and yeast extract were added to the basal medium B (for its composition, see Table 2) to prepare a serum-free medium.
  • the total amount of the soybean protein hydrolysate and the yeast extract added was 5 g/liter. Based on the results of Example 1 above, the ratio (by weight) of the amount of addition of soybean protein hydrolysate to the amount of addition of yeast extract was selected at 60:40. To this composition, wheat protein hydrolysate was further added. Using the resultant medium, cell count on day 4 of the cultivation and thereafter, cell viability from day 4 to day 7, and the total amount of the recombinant protein accumulated in the medium up to day 7 were compared.
  • Cultivation conditions were as follows. Thirty milliliters of the medium was placed in a 125-ml flask. Cells were seeded at a density of 3.0 x 10 5 cells/ml and cultured under agitation at 160 rpm, at 37 °C , under 5% C0 2 . The results, i.e., cell count on day 4 of the cultivation and thereafter, cell viability from day 4 to day 7, and the total amount of the recombinant protein accumulated in the medium up to day 7 are shown in Figs. 4, 5 and 6, respectively.
  • the serum-free medium used for the cultivation had the same composition as basal medium composition A shown in Table 3 except for the amount of addition of a recombinant human insulin.
  • Basal medium composition A shown in Table 3 except for the amount of addition of a recombinant human insulin.
  • 3 g/liter of soybean protein hydrolysate, 2 g/liter of yeast extract and 1 g/liter of wheat protein hydrolysate were added.
  • a recombinant human insulin was added to the medium at 5 mg/ liter, 2.5 mg/liter, 1.25 mg/ liter or 0 mg/liter (no addition).
  • the cultivation condition for each case was as described m Example 2 above.
  • CHO cells were seeded in each serum-free medium at a density of 3.0 x 10 5 cells/ml.
  • CHO cells were cultured in advance in a serum-free medium (basal medium A supplemented with 3 g/liter of soybean protein hydrolysate, 2 g/liter of yeast extract and 1 g/ liter of wheat protein hydrolysate) under the conditions described in Example 2 above.
  • a serum-free medium basal medium A supplemented with 3 g/liter of soybean protein hydrolysate, 2 g/liter of yeast extract and 1 g/ liter of wheat protein hydrolysate
  • Panel “a” in Fig. 7 shows the time course of the number of cultured cells for 8 days from the beginning of the cultivation
  • panel “a” in Fig. 8 shows the time course of the total yield of the recombinant protein produced by the cultured cells during the above period; in each panel , 4 different conditions were used as regards the amount of addition of the recombinant human insulin.
  • Panel “b” in Fig. 7 and panel “b” in Fig. 8 show the further results obtained when the cells cultured for 8 days were subcultured in a fresh medium.
  • Example 2 In the serum-f ree medium of Example 2 which was obtained by adding 3 g/ liter of soybean protein hydrolysate, 2 a/ liter of yeast extract and 1 g /liter of wheat protein hydrolysate to the basal medium shown in Table 2, 8 g/ liter of glucose was replaced with 1.6 g/liter of glucose and 6.4 g /liter of fructose.
  • the initial medium containing 8 g/ liter of glucose and tr.e newly prepared medium containing 1.6 g/ liter of glucose and 6.4 g /liter of fructose
  • two other media were prepared by increasing the amount of addition of the recombinant human insulin in the said respective media from 5 mg/liter to 10 mg/ liter.
  • transformed CHO cells were cultured. Cultivation conditions were as follows. Thirty milliliters of the medium was placed a 125-ml flask. Cells were seeded at a density of 3.0 x 10 5 cells/ml and cultured under agitation at 160 rpm, at 37 °C , under 5% C0 2 .
  • Fig. 9 shows the results of comparison of viabilities on day 4 of the cultivation and thereafter.
  • Fig. 10 shows the results of comparison of the total yields cf the recombinant protein by the transformed CHO cells up to day 7 of the cultivation. As shown in Fig.
  • t was found that the viability of the cultured cells on day 4 of the cultivation and thereafter significantly decreases as a result of the substitution of fructose for glucose. It was found that this decrease in the viability of the cultured cells occurred concomitantly with the increase in the total yield of the recombinant protein by the transformed CHO cells. Further, it was confirmed separately that as the ratio of substitution of fructose for glucose increases, the above-mentioned influence increases in proportion.
  • the viability of cultured cells can be maintained at 20% or highed by adjusting the ratio of substitution of fructose for glucose to less than 80%, although the decrease in the viability of cultured cells is not necessarily desirable when transformed CHO cells are subcultured in a fresh medium for continuous production of a recombinant protein.
  • the decrease in viability will not give an extremely bad influence and will not lead to an overall decrease in productivity in the continuous production of a recombinant protein by subculture.
  • the serum-free medium of the invention was compared with a serum- free medium containing animal-derived proteins which is obtained by replacing the three characteristic components of the serum-free medium of the invention (i.e., soybean protein hydrolysate, yeast extract and wheat protein hydrolysate) with animal-derived proteins that are commonly used as additives to a serum-free medium. It was demonstrated that there is no significant difference between these two serum-free media in cultivation results, and that the addition of the above three characteristic components in the serum-free medium of the invention produces a superior or at least equivalent effect compared to the addition of animal-derived proteins which are commonly used additives .
  • the three characteristic components of the serum-free medium of the invention i.e., soybean protein hydrolysate, yeast extract and wheat protein hydrolysate
  • basal medium C shown in Table 4 below and supplemented with bovine serum albumin, bovine fetuin and primatone (beef hydrolysate) was used.
  • the bovine serum albumin used was a commercial product verified to be free of contamination with virus and others after inspection of contaminants such as virus .
  • the bovine fetuin and the primatone used were also commercial products verified to be free of contamination with virus and others after inspection of contaminants such as virus.
  • the amounts of addition of the respective animal- derived proteins per liter of the medium were as follows: bovine serum albumin (virus free) 100 mg, fetuin 200 mg, human transferrin 5 mg and primatone 2500 mg. Table 4.
  • Basal Medium Composition C shown in Table 4 below and supplemented with bovine serum albumin, bovine fetuin and primatone (beef hydrolysate) was used.
  • the bovine serum albumin used was a commercial product verified to be free of contamination with virus and others after inspection of contaminants such as virus .
  • an iron-EDTA complex is also used as an iron source addition to an inorganic iron salt.
  • the amount of addition of this complex represents the final concentration in the medium when the preparation of the medium has been completed.
  • the serum-free medium containing the three components, soybean protein nydrolysate, yeast extract and wheat protein hydrolysate is basal medium A shown in Table 1 and supplemented with 3 g/ liter of soybean protein hydrolysate, 2 g/liter of yeast extract and 1 g/liter of wheat protein hydrolysate. Further, as in Example 4, 8000 mg/liter of glucose m the composition of Table 1 was replaced with 1400 mg/liter of glucose and 6600 mg/liter of fructose. To the resultant basal medium, 3 g/ liter of soybean protein hydrolysate, 2 g/ liter of yeast extract and 1 g/liter of wheat protein hydrolysate were added, and the resultant medium was used for comparison.
  • Another medium was prepared by eliminating only glutamme from the above-mentioned fructose-contaming medium and used for comparison.
  • transformed CHO cells were cultured. Cultivation conditions were as follows. Thirty milliliters of the medium was placed in a 125 ml flask. Cells were seeded at a density of 3.0 x 10 5 cells/ml and cultured under agitation at 160 rpm, at 37 °C , under 5% C0 2 .
  • the serum-free medium containing animal-derived proteins and the serum-free medium which is the basal medium of Table 1 plus the three components, soybean protein hydrolysate, yeast extract and wheat protein hydrolysate; as shown in Fig. 11, the two serum-free media exhibited comparable results as regards the total yield of the recombinant protein accumulated in the medium up to day 7. Similarly, there is only a small difference between these two media in cell viability from day 3 to day 7 of the cultivation, as shown in Fig. 12.
  • the serum-free medium containing the three components, soybean protein hydrolysate, yeast extract and wheat protein hydrolysate produces favorable results almost equal to those produced by the serum-free medium containing three animal-derived proteins, bovine serum albumin, bovine fetuin and primatone (beef hydrolysate), in terms of cultivation rate and production yield of the recombinant protein.
  • the serum-free medium of the invention which is obtained by adding soybean protein hydrolysate and yeast extract to a basal medium containing no components separated from animals promotes and stabilizes the cell growth of transformed CHO cells or the like as induced by a recombinant growth factor added to the serum-free medium, whereby the cultivation rate can be maintained at high level. Further, the serum-free medium of the invention can increase the yield of a recombinant protein produced by the cultured cells .
  • the serum-f ree medium of the invention has the advantage that by adding wheat protein hydrolysate , as we l l as soybean protein hydrolysate and yeast extract , the cel l viability can be maintained at high level so that when the cultured cells are subcultured , the cultivation efficiency and the total yield of the recombinant protein produced by the cultured cells are stabilized in each generation .
  • a recombinant protein of interest can be produced consistently at high efficiency and with good reproducibility by applying the method of growing transformed animal cells in the serum-free medium of the invention.
  • the serum-f ree medium of the present invention can achieve an ability comparable to that of a conventional serum-containing medium in terms of culturing animal cel ls and is therefore usef l .
  • the serum-free medium of the present invention can be used to culture desired animal cells and to produce desired recombinant proteins .

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Abstract

La présente invention concerne un milieu exempt de sérum permettant de mettre en culture des cellules animales, ce milieu contenant un hydrolysat protéique de soja et un extrait de levure. Cette invention concerne également un procédé de mise en culture de cellules animales, ce procédé consistant à mettre en culture des cellules animales dans ledit milieu exempt de sérum. Cette invention concerne enfin un procédé de production d'une substance, ce procédé consistant à mettre en culture des cellules animales dans ledit milieu exempt de sérum, à amener une substance à être produite et sécrétée par ces cellules animales, et à isoler cette substance du milieu exempt de sérum.
PCT/JP1999/003735 1998-07-10 1999-07-09 Milieu exempt de serum pour mettre en culture des cellules animales WO2000003000A2 (fr)

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JP2000559222A JP3934877B2 (ja) 1998-07-10 1999-07-09 動物細胞培養用無血清培地

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US6406909B1 (en) 2002-06-18
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